College of Optometry, University of Houston, Houston, Texas, USA.
Department of Biology/Chemistry, iBiOs-Integrated Bioimaging Facility, CellNanOs - Center of Cellular Nanoanalytics, Osnabrück, Germany; Department of Microbiology, University of Osnabrück, Osnabrück, Germany.
J Biol Chem. 2023 Nov;299(11):105282. doi: 10.1016/j.jbc.2023.105282. Epub 2023 Sep 22.
The intracellular domains of connexins are essential for the assembly of gap junctions. For connexin 36 (Cx36), the major neuronal connexin, it has been shown that a dysfunctional PDZ-binding motif interferes with electrical synapse formation. However, it is still unknown how this motif coordinates the transport of Cx36. In the present study, we characterize a phenotype of Cx36 mutants that lack a functional PDZ-binding motif using HEK293T cells as an expression system. We provide evidence that an intact PDZ-binding motif is critical for proper endoplasmic reticulum (ER) export of Cx36. Removing the PDZ-binding motif of Cx36 results in ER retention and the formation of multimembrane vesicles containing gap junction-like connexin aggregates. Using a combination of site-directed mutagenesis and electron micrographs, we reveal that these vesicles consist of Cx36 channels that docked prematurely in the ER. Our data suggest a model in which ER-retained Cx36 channels reshape the ER membrane into concentric whorls that are released into the cytoplasm.
连接子的细胞内结构域对于间隙连接的组装是必不可少的。对于主要的神经元连接子连接蛋白 36（Cx36），已经表明功能失调的 PDZ 结合基序会干扰电突触的形成。然而，目前尚不清楚该基序如何协调 Cx36 的运输。在本研究中，我们使用 HEK293T 细胞作为表达系统，对缺乏功能性 PDZ 结合基序的 Cx36 突变体进行了表型特征分析。我们提供的证据表明，完整的 PDZ 结合基序对于 Cx36 的正确内质网（ER）输出至关重要。去除 Cx36 的 PDZ 结合基序会导致 ER 滞留，并形成含有间隙连接样连接子聚集物的多膜囊泡。通过定点突变和电子显微镜的组合使用，我们揭示了这些囊泡由过早停靠在内质网中的 Cx36 通道组成。我们的数据表明了一种模型，即 ER 滞留的 Cx36 通道将 ER 膜重塑成同心的漩涡，然后释放到细胞质中。
